The proposed research is focused on the development of high frequency dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) as an approach to enhance sensitivity in solid state and solution NMR based structural biology experiments. In addition, we plan time domain EPR spectroscopy to provide support for the DNP research and because the spectra are inherently interesting in themselves. We plan to address structural questions in a number of interesting biochemical systems containing intrinsic or extrinsic paramagnetic centers. 1 Polarizing Agents: We plan to continue to develop new polarizing agents with studies of: (1) bis-TEMPO-bis-ketal with fixed relative orientations of the TEMPO's;(2) TEMPO tethered to a water soluble BDPA radical to satisfy the matching condition for the cross effect [[unreadable]2e- [unreadable]1e = [unreadable]n];(3) a series of biradicals that provide strong [0.6-1.5 GHz] electron-electron dipole couplings. 2 Time Domain DNP Experiments CW DNP enhancements mechanisms exhibit a [unreadable]0-1 or [unreadable]0-2 dependence, but pulsed DNP mechanisms do not. For this reason we will be investigating the integrated solid effect (ISE), versions of electron-nuclear Hartmann-Hahn cross polarization (eNCP), and RF-DNP. We plan to perform these experiments at 9 GHz, where it is easy to control microwave pulses and phases, as well as at 140 GHz using existing equipment and a gyroamplifier under development. 3 Solution DNP Experiments : Temperature jump DNP (TJ-DNP) where we polarize the sample at 90 K, melt it rapidly with a CO2 laser, and acquire the solution state NMR spectrum. TJ-DNP yields 2D 13C-13C solution spectra with a factor of 100-170 signal enhancement over the liquid state spectrum at 300 K. We plan to refine the TJ-DNP protocol to include lower temperatures, single scan protocols, and acquisition of multidimensional 13C-15N spectra. 4 Applications to Proteins We plan to optimize the signal enhancements and the quality of the available structural information for membrane and amyloid proteins. We plan to use 2H labeled Pf1 phage coat protein and U-(13C,15N) GB1 to optimize the enhancements. We will be extending the experiments to 700 MHz (1H frequency) using a tunable 460 GHz gyrotron that recently operated CW for 2-3 day periods.